PONE-D-21-08289

Unraveling the unbinding pathways of SARS-CoV-2 Papain-like proteinase known inhibitors by Supervised Molecular dynamics simulation

PLOS ONE

Dear Dr. Aryapour,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address each of the points raised by the reviewers and note in the cover letter the corresponding revisions of the manuscript. Include in the cover letter detailed responses to the comments of the reviewers.

Please submit your revised manuscript by May 29 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the reviewers. You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Emilio Gallicchio, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.  

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript
• A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)
• A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments for the Authors

The outbreak and spread of COVID-19 diseases caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection now is well-known as a global concern to the public health worldwide. Most infected people develop asymptomatic symptoms or mild to moderate illness and recover without hospitalization. However, for these of severe cases will have worsening dyspnea accompanied by hypoxemia and further turn to progressive respiratory failure, which can be lethal. Vaccine can prevent the spread of COVID-19 for the uninfected individuals. However, for these SARS-CoV-2 infected patients with acute diseases, pharmaceutic intervention with significant therapeutic efficiency would be an essential antiviral treatment strategy. The papain-like protease (PLpro) of SARS-CoV-2 is a highly versatile enzyme that processes viral polyproteins to generate a functional replicase complex and enable viral spread, also essential for cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses. Inhibition of SARS-CoV-2 PLpro with inhibitor could be a potential therapeutic strategy through impeding viral replication in infected cells. In this study, Farzin et al. performed Supervised Molecular Dynamics (SuMD) simulations to decipher the unbinding pathway of SARS-CoV-2 PLpro and its four inhibitors including GRL0617 and its derivates. The simulation stability of SARS-CoV-2 PLpro and GRL0617 outperforms those of the other three, in agreement with the experimental IC50 data. The 2D free energy changes caused by the binding of inhibitors were calculated. Their respective intermediate binding poses, protein-ligand interactions such as hydrogen bond changes over time, RMSF of PLpro residues at holo state were analyzed. In summary, the work is perspectively important and properly descripted. It provides useful conclusions about how GRL0617 and its derivates disassociated with the PLpro binding, in particular, the major role of Tyr268 and Gln269 residues of the BL2 loop in the unbinding pathways.

There are several points described below,

1.For each inhibitor, 3 series of SuMD simulations were performed. However, the 2D free energy profiles are likely not converged, as new minima emerged in each of three maps, for instance, Fig. 5h-j. More series of SuMD simulations should be needed.

2. Although the authors mentioned binding free energy calculation by MM/PBSA method, however, I did not find the calculation results. Also, the authors also estimated the contribution of each amino acids, as shown in Supplementary Figs. 2-3, however, the authors did not describe in the manuscript about how these contributions were estimated, by free energy decomposition method?

3.I suggest to highlight the catalytic triad and the BL2 loop with different color in the upper panel of Fig. 1, while yellow colored residues interacting with GRL0617 ligand should be labeled too in the lower panel of Fig. 1. In addition, BL2 loop should be defined, from which residue to which residue?

4.(1)What is the reference structure used for RMSD calculation in Figs. 2-4a?

(2)I suggest to split the total interaction so as to also include the ele and vdw interactions in Figs. 2-4c to show their changes.

(3)The distance of hydrogen bonds in Figs. 2-4 should be labeled.

(4)There are many intermediate states during a MD simulation, which frames were extracted as Figs. 2-4e,f? Please clarify. It is important to find these key intermediate states correctly for each inhibitor, so that their comparisons were fair.

(5)Did the author extract out the structures from the minima of three free energy landscapes in Figs. 2-4h-j? These metastable structures should be compared and discussed.

(6)For each inhibitor, did three series SuMD show the same trajectory path line? I suggest the authors provide three trajectory path lines with three colors, to see if the inhibitor exit the binding pocket with different pathways.

(7)The crystallographic (native) conformation should be marked in Fig.s 2-4h-j.

5.For PLP_Snyder441-Plpro complex, the authors mentioned that “In all of the three replicas performed, it was observed that the movements of the BL2 loop had a direct effect on the inhibitors (Fig. 3a).” However, as shown in Fig. 3a, the replica 3 seems an opposite case. Because although BL2 dramatically shifted at ~8 ns, PLP_Snyder441 looked stable over the whole MD time. The reason should be tracked by analyzing the extracted structures.

6.The authors should run a direct MD simulation for SARS-CoV-2 PLpro at apo state, and calculate RMSF for each residue to show the nature flexibility particularly for BL2 loop to support the point that “The presence of the ligand in the active site may probable lower these fluctuations”.

7.In Fig. 6, the binding ligands of crytal structure (green) and MD structure (black) should be presented using the same color with their corresponding proteins.

Minor issues:

1. “The RMSD values of the inhibitor throughout the simulations (Fig. 2a) indicated that it only takes very short amounts of time for the inhibitor to get from the crystallographic conformation to the unbound state”, the “amounts” here is subscript, please correct.

2. Please provide legends for movies as well. The movies should be cited in the main text properly.

3. “On the other hand, these extra hydrogen bonds can even force the inhibitor to lift up and in the meanwhile the interactions can get water-mediated (Fig. 4c).” “Fig. 4c” should be “Fig. 4e”.

Reviewer #2: The article is straightforward and for most of the parts clear. The impact that it would provide is significant since it provides insight into potential drug discovery against COVID-19. I therefore believe that it is valuable, and it should be published. However, it needs some revisions:

1. In Table 1 the authors claim “In terms of duration, the more potent GRL0617 showed better performance than the others, except for the PLP_Snyder530, and the already published activity assays also suggest the same. However, comparing the duration times of the unbinding events is not a correct way to compare the potency of the inhibitors.”

If “comparing the duration times of the unbinding events is not a correct way to compare the potency of the inhibitors”, what is the point of even including Table 1?

2. In figure 1, none of the ligand-receptor interactions (H-bonds and pi-stacking) are depicted.

3. In all the figures, I notice inconsistencies: the BL2 is not always labelled, some key residues are not always labelled, pi-stacking interactions are never depicted.

4. In the last portion of the discussion I notice another contradiction. The authors claim: “Among the synthesized inhibitors by Osipiuk et al.[23], PLP_Snyder495 had extra groups on the benzamide moiety that could make strong hydrogen bonds with the Glu167 which is not on the BL2 loop. However, this residue is fully exposed to water molecules and the hydrogen bonds can easily get water-mediated and broken and as a result, this compound had the worst performance in our simulations and left the binding pocket in very short amounts of time (Table 1). We believe that minimizing interactions with the BL2 loop and maximizing interactions with the inner parts of the binding pocket where there is more limited access to water molecules is the best way to improve these inhibitors.”

Glu 167 seems to be in the inner part of the pocket and yet the molecule does not bind strongly enough. This seems to contradict the last sentence. Can you please explain?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Pierpaolo Cordone

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Unraveling the unbinding pathways of SARS-CoV-2 Papain-like proteinase known inhibitors by Supervised Molecular Dynamics simulation

PONE-D-21-08289R1

Dear Dr. Aryapour,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Emilio Gallicchio, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: